Methods to regulate polarization and enhance function of excitable cells

ABSTRACT

Minimally invasive delivery with intercellular and/or intracellular localization of nano- and micro-particle solar cells within and among excitable biological cells to controllably regulate membrane polarization and enhance function of such cells. The cells include retinal and other excitable cells.

This application is a continuation in part of U.S. patent applicationSer. No. 13/367,984 filed Feb. 7, 2012, now U.S. Pat. No. 8,460,351;which is a continuation-in-part of U.S. patent application Ser. No.13/088,730 filed Apr. 18, 2011, now U.S. Pat. No. 8,409,263; which is acontinuation-in-part of U.S. patent application Ser. No. 11/197,869filed Aug. 5, 2005, now U.S. Pat. No. 8,388,668; each of which isexpressly incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to combined methods to regulate polarization andenhance function of excitable cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of a longitudinal section of a human eye.

FIG. 2 is an enlarged diagrammatic illustration of the circled area 2 ofFIG. 1 showing detailed retinal structures.

FIG. 3 shows the eye of FIG. 1 with a cannula delivering particles tothe retina in accordance with one embodiment of the invention.

FIG. 4 is an enlarged diagrammatic illustration of the circled area 4 inFIG. 3 showing particles jetting from a cannula and dispersingthroughout retinal structures.

DETAILED DESCRIPTION

Combination mechanisms to correct, reduce, and/or prevent physiologicalelectro-sensory damage or electromotor damage and promote functionalrecovery of excitable cells, e.g., neurons in the central nervous system(i.e., brain and spinal cord) and neuronal cells involved with visual,auditory, vocal, olfactory responses, e.g., retinal cells in the eye,cochlear cells in the ear, olfactory cells in the nose, etc., andneurons in the peripheral nervous system are provided. The inventivecombination methods can be thought of as akin to combination approachesin treating neoplastic lesions, but targeting less thanoptimally-functioning excitable cells.

In one embodiment, the combined method promotes functional recovery andcontrollably regulates plasma membrane polarization of a functionalexcitable neuronal cell. A biomolecule effecting gene therapy isadministered into an eye and/or central nervous system of a patient inneed of the therapy (e.g., a patient with a neuronal disease). Quantumdots are administered into the eye and/or central nervous system of thepatient, either simultaneously or sequentially either before or afterthe biomolecule is administered. Light is applied to the eye or centralnervous system to controllably activate the quantum dots by controllingexposure time, exposure intensity, exposure site, etc. to controllablyregulate the plasma membrane polarization of the functional excitableneuronal cells and to provide the biomolecule to the neuronal cells. Inone embodiment, the biomolecule is directly or indirectly associatedwith, or covalently conjugated to, the quantum dots so that in a singleadministration (e.g., one injection), both biomolecule and quantum dotscomponents are provided to the patient. Once administered, the quantumdots can be imaged, tracked, monitoring, evaluated in the patient usinga sensor or other tracking agent using methods well known in the art(e.g., digital imaging, etc.).

The light sensitive molecules may be provided to specific neurons fortherapy. As one example, they may be provided to an optic nerve forretinal therapy. As another example, they may be provided to anolfactory nerve for nasal nerve therapy, and/or as an point of entry forbrain therapy, etc. As another example, they may be provided toselective or non-selective sites for selective stimulation of variousregions, either alone or in combination. As non-limiting examples ofselective stimulation of central nervous system nerves, the visualcortex can be stimulated through specific light stimulation of theretina, the olfactory neuron can be stimulated by smell, the auditoryneuron can be stimulation by sound, etc. As non-limiting examples ofselective stimulation of peripheral nervous system nerves, chronic painmay be controlled by direct stimulation of the appropriate nerves, andappetite may be suppressed by direct stimulation of appropriate nerves.

Stimulation by light may be achieved by several mechanisms, as known toone skilled in the art. For example, using activation of quantum dots inthe brain as an exemplary, non-limiting example, activation may beprovided by a fiber optic device surgically placed at the desired areaof the brain, located under the scalp, and illuminated by a lightsource, e.g., a light emitting diode (LED) through a small window madein the skull replaced by clear glass at a desired area. Such a windowmay remain hidden under the skin, because it is known that light canpenetrate a few millimeters into skin. An analogous concept may be usedfor stimulating other areas of the central nervous system, theperipheral nervous system, or heart or other muscles, with or withoutapplication of a fiber optic device if quantum dots are injected throughan opening into the superficial area of the brain, nerve, heart muscle,etc. Such stimulation may controllably regulate, i.e.,activate/deactivate, by using an appropriate wavelength of light, withor without a processor with the specific neuronal code as pulses.Quantum dots may be used in conjunction with stem cell therapy or inconjunction with other devices, e.g., prosthetic devices, that areactivated or otherwise rely or light and/or electrical current.

In addition to using the method for the above indications and fortreatment of retinal degeneration, etc. and posttraumatic epilepsy, themethod also has applications in amelioration of the underlying pathologyand/or symptoms of genetic and/or degenerative diseases, e.g., retinitispigmentosa, retinal degeneration, central nervous system pathologiessuch as Alzheimer's disease and Parkinson disease, dopamine-regulateddisorders such as migraines, autism, mood disorders, schizophrenia,senile dementia, sleep disorders, restless leg syndrome, and depression.Tourette syndrome, restless leg syndrome, and stuttering are a part ofthe same spectrum of diseases characterized by malfunctioning membranepotential and electrical pulse transmission. The consequences ofinfectious diseases, epilepsy, paralysis, and traumatic injury of thebrain and/or peripheral nerves are also amenable to therapy with theinventive method. All such disorders can be influenced either withquantum dot administration alone or with medication modifying cellmembrane potential, e.g., carbonic anhydrase inhibitors. Ameliorationincludes any reduction in the signs, symptoms, and/or etiology,including but not limited to prevention, therapy, and curative effects,of any of the above indications. As one example, quantum dots may betargeted to dopamine-regulated nerves for therapy of migraines, mooddisorders, etc. As another example, quantum dots can be used for deepsubthalamic, cerebral, or cortical and peripheral nerve stimulation fortherapy of Parkinson's disease, etc.

A viral vector (e.g., adenovirus, adeno-associated virus, retrovirus)can provide the biomolecule, which can be a natural or syntheticprotein, peptide, nucleic acid, oligonucleotide, etc. when conjugatedwith quantum dots. In one embodiment, the biomolecule is a cell membranechannel protein. If the same wavelength of light stimulates both quantumdots and protein (or other biomolecule), the result is an enhancedaction potential in the excitable cells, i.e., this embodiment achievesa synergistic effect. If a different wavelength of light stimulates thequantum dots and protein (or other biomolecule), the result is asubsequent action potential in the excitable cells, i.e., thisembodiment achieves silencing of the action potential in the cell. Inboth cases, the “tunable” selection of the biomolecule and the quantumdots, as well as the specific excitation energy (typically light butalso ultrasound radiation energy can be used) applied, provides acontrolled and regulated process. In turn, the high degree of controlenhances efficacy and safety and permits close monitoring andregulation.

Delivery and intercellular and/or intracellular localization of nano-and micro-particle solar cells within and/or among excitable biologicalcells to regulate membrane polarization of biological cells combinedwith other methods to promote functional recovery of damaged excitablecells in the eye and central nervous system. The inventive methodprovides solar cells in a minimally invasive procedure into the eye,heart, and/or the central nervous system; the solar cells are notimplanted in the body in an invasive procedure. The inventive methodprovides a plurality of solar cells as discrete individual particles;the solar cells are not connected as a unit and do not have a backinglayer or backing material. The inventive method uses solar cells thatmay be activated by ambient light; the method does not use an electricalapparatus and thus does not use photodiodes, stimulating electrodes, orother electrical devices. The inventive method uses solar cells toenhance the regulation of polarization by the excitable biological cellsthemselves; the solar cells facilitate or boost the ability of excitablebiological cells to normalize or regulate their own polarity. Theinventive method provides for excitable biological cells to regulatetheir own polarity; stimulation of the solar cells used in the inventiondoes not generate an action potential to regulate polarity, but insteadfacilitates the biological cells themselves to regulate polarity. Theinventive method provides quantum dots in combination with therapies toenhance functional recovery of neuronal cells damaged by differentetiologies, including genetic disorders, ischemic or vascular damage,and age-related damage. By combining modulation of cell polarization,which takes advantage of the ability to regulate quantum dots, withgenetic and other approaches to therapy, neuronal degenerative processare ameliorated.

Biological cells are bound by a plasma membrane. In all cells, thismembrane has a resting potential. The resting potential is an electricalcharge across the plasma membrane of the non-excited or resting cell,rendering the interior of the cell negative with respect to theexterior. Hence, the plasma membrane of all biological cells in theirresting state is polarized.

The extent of the resting potential varies among different cell types.In cells such as nerve, muscle, and retinal cells, which are excitablein that they can be stimulated to create an electric current, theresting potential is about −70 millivolts (mv). This resting potentialarises from two components of the plasma membrane: the sodium/potassiumATPase, which pumps two potassium ions (K⁺) into the cell for everythree sodium ions (Na⁺) it pumps out of the cell, and “leakiness” ofsome K⁺ channels, allowing slow facilitated diffusion of K⁺ out of thecell. The result is a net loss of positive charge from within theresting cell.

Certain external stimuli reduce the charge across the plasma membrane,resulting in membrane depolarization. As one example, mechanical stimuli(e.g., stretching, sound waves) activate mechanically-gated Na⁺channels. As another example, certain neurotransmitters (e.g.,acetylcholine) open ligand-gated Na⁺ channels. In each case, thefacilitated diffusion of Na⁺ into the cell depolarizes the membrane; itreduces the resting potential at that membrane location. This creates anexcitatory postsynaptic potential (EPSP).

If the potential at any membrane location is reduced to the thresholdvoltage, many voltage-gated Na⁺ channels open in that location,generating an influx of Na⁺. This localized, sudden, completedepolarization opens adjacent voltage-gated Na⁺ channels. The result isa wave of depolarization along the cell membrane, referred to as theaction potential or, in excitable cells, an impulse.

A second stimulus applied to an excitable cell within a short time (lessthan 0.001 second) after the first stimulus will not trigger anotherimpulse. This is because the membrane is depolarized, leaving the cellin a refractory period. Only when the −70 mv polarity is reestablished,termed repolarization, will an excitable cell be able to respond toanother stimulus. Repolarization is established by facilitated diffusionof K⁺ out of the cell. When the cell is finally rested, Na⁺ that enteredthe cell at each impulse are actively transported back out of the cell.

Hyperpolarization occurs when negatively charged chloride ions (Cl⁻)enter the cell and K⁺ exit the cell. Some neurotransmitters mayfacilitate this by opening Cl⁻ and/or K⁺ channels in the plasmamembrane. Hyperpolarization results in an inhibitory postsynapticpotential (IPSP); although the threshold voltage of the cell isunchanged, it requires a stronger excitatory stimulus to reachthreshold.

Abnormal cell polarization may affect regenerative and/or functionalprocess of excitable cells, and result in cell dysfunction. Abnormalcell polarization includes, but is not limited to, any of the followingand whether transient or sustained: loss of polarization, decreasedpolarization, altered polarization, hyperpolarization, and any deviationfrom normal cell polarization. Excitable cells include, but are notlimited to, sensory cells (e.g., retina and macula of the eye), neuronalcells in the central nervous system (CNS) (brain and spinal cord) andperipheral nervous system, muscle cells (striated, cardiac, and smoothmuscle cells).

The orientation of the cell with respect to its apical, lateral, andbasal surfaces may affect polarization and may be regulated by theinventive method. Adjacent cells communicate in the lateral domain, withattachment or contact sites by which cells adhere to one another.Terminal bars, attachment sites between cells that act as a barrier topassage of substances, are located around the entire circumference ofcells and are composed of junctional complexes responsible for joiningindividual cells. Occluding junctions, also referred to as tightjunctions or zonula occludentes, are located apically within the lateraldomain and encircle the cell, separating the luminal region from theintercellular space and cytoplasm. These are narrow regions of contactbetween the plasma membranes of adjacent cells and seal off theintercellular space, forming an impermeable diffusion barrier betweencells and preventing proteins from migrating between apical and lateralsurfaces of the cell. In one embodiment, the method selectivelyregulates polarization in areas of the cell bound by occludingjunctions. Particles may be selectively positioned and/or selectivelyregulated to regulate polarization at a desired site.

Ischemic cell death is caused by failure of the ionic pumps of theplasma membrane. Depolarization of the plasma membrane in retinal cellsand subsequent synaptic release of L-glutamate are implicated inischemic retinal damage. Mali et al. (Investigative Ophthalmology andVisual Science, 2005, 46, 2125) reported that when KCI, a known membranedepolarizing agent, is injected into the vitreous humor, the subsequentmembrane depolarization results in a dose- and time-related upregulationof matrix metalloproteinase (MMP)-9 activity and protein in the retina.This was associated with an increase in proapoptotic protein Bax andapoptotic death of cells in the ganglion cell layer and inner nuclearlayer, and subsequent loss of NF-L-positive ganglion cells andcalretinin-positive amacrine cells. A synthetic MMP inhibitor inhibitedKCI-mediated MMP-9 upregulation, which led to a significant attenuationof KCI-induced retinal damage. Regulating polarization thus inhibitsMMP-9 and decreases damage that can diminish visual acuity.

Methods to regulate membrane polarization of excitable cells assist inminimizing physiologic damage and reducing pathology including but notlimited to ischemic damage to the retina, degenerative diseases of theretina including but not limited to retinitis pigmentosa, ischemicand/or degenerative diseases of cardiac muscle, and/or ischemic anddegenerative diseases of cerebral tissue, etc. In turn, the methodminimizes or prevents undesirable effects such as loss of visual acuity,myocardial infarction, cerebral stroke, etc. and enhances a patient'squality of life.

The inventive method may be more fully appreciated with respect to itsutility in a single organ, such as the eye. One skilled in the art willrealize, however, that it is not so limited and is applicable to otherexcitable cells.

In one embodiment, the inventive method externally administers to apatient a composition or, alternatively a device in a biocompatiblecomposition, comprising quantum dots or solar cells to stimulate thecell membranes from inside of the cell or outside of the cell of allretinal cells. In one embodiment, the quantum dots injected into the eyeand are delivered to the retinal cell cytoplasm or nucleus. In oneembodiment, the quantum dots are introduced into the central nervoussystem. In one embodiment, the quantum dots are conjugated or otherwiseassociated with proteins or other moieties and provided using a vectorto a patient to effect functional recovery of neuronal cells. Onenon-limiting example of this embodiment is quantum dots conjugated witha channel proteins introduced via a viral vector (e.g., adeno-associatedvirus (AAV)) to effect retinal gene therapy. Such a vector and/orquantum dots can be labeled for visualization, tracking, sensing, etc.For example, the quantum dots can be labeled or tagged with a signalrecognition moiety. Such a vector can incorporate quantum dots into theviral capsid using, e.g., (poly)ethylene glycol (PEG) moieties. Anothernon-limiting example is the use and selective regulation, selectiveactivation/deactivation alone or in combination, to monitor interferingRNA (RNAi) delivery and regulate gene silencing. Another non-limitingexample is the use of quantum dots for in situ visualization of geneexpression. This may be performed using quantum dot-DNA-coated polymer.Combinations of these embodiments are contemplated and included, usingmethods known by one skilled in the art and as subsequently described.

As used herein, particles, quantum dots, and solar cells are usedsynonymously.

The retinal cells comprise at least ganglion cells, glial cells,photoreceptor cells, Muller cells, bipolar cells, horizontal cells,microglial cells, and cells of the neural fibers, etc. The amount ofstimulation, or degree of membrane stimulation, can be regulated by theamount of energy provided by the particles. The total amount of energyprovided by the particles to transmit to the membrane depends upon thetime of particle activation.

The particles are activated by the energy source; the response to thespecific wavelength depends on the inner material building the innersemiconductor. The energy source to activate the particles providesambient light, ultraviolet light, visible light, infrared light, orultrasound radiation. In one embodiment, the particles respond to blue,red, green, or IR light. In one embodiment, a plurality of particlesrespond to various specific wavelengths. In one embodiment, theparticles have multiple semiconductor cores, and thus respond to variouswavelengths. The wavelength selections are photons with differentenergies. The particles must have energy bandgaps or energy statues thatmatch the energy of the photons. One skilled in the art tunes the energylevels using materials with different band-gaps or by carefullyselecting the quantum size as it effects the energy level. Thus, oneuses different size particles and/or particles with different cores. Inone embodiment, the activation time interval ranges from 1 nanosecond to100 nanoseconds. In one embodiment, the activation time interval rangesfrom 1 second to 100 seconds.

The source of energy activates the particles for the particles toprovide sufficient energy to activate the membrane. In one embodiment,the energy source sufficient to activate the particles ranges from aboutone picojoule to one microjoule. In one embodiment, the activationenergy source is external ambient light. In one embodiment, theactivation energy source is a diode, LED, etc. Other activation energysources are possible, as known by one skilled in the art. The energysource provides electromagnetic radiation, as known to one skilled inthe art. Electromagnetic radiation includes infrared radiation (700 nmto 1 mm), visible light (380 nm to 760 nm), and ultraviolet radiation (4nm to 400 nm). The energy source is varied to vary the response of theparticles; as one skilled in the art is aware, the shorter thewavelength, the more energy is delivered. As an example, infraredwavelengths (thermal activation), visible and ultraviolet wavelengthsare provided for activating the particles to produce the desiredphotovoltaic (energy) response from the particle by a separate energysource or one that can provide combinations of the required wavelengthranges. The energy source(s) may be externally programmed (such as bycomputer software) to produce different wavelengths resulting inphotovoltaic responses at desired time intervals. The regulation orcontrol of the timed production of generated photovoltaic responses fromthe particles can be used to control the regulation of cell membranepotentials. The energy input from the energy source may be varied tovary the particles responses, hence regulating and/or controlling themembrane potential. The particles respond to the specific wavelength(s)to which they are exposed. A specific coating to the particles rendersthem specific. The protein coating can direct them to attach to certaincell membranes, and/or to enter a cell such as a normal cell, a tumorcell, a nerve cell, a glial cell, The particles, albeit relativelynon-selective, can potentially increase the membrane potential of anycells to which they come into contact. After exposure to light, a diode,etc. they emit an electrical potential, current, or fluorescence. Theelectrical potential generated by this exposure to radiation increasesthe cell membrane potential.

FIG. 1 shows a mammalian eye 10. The structures and locations of theanterior chamber 11, cornea 12, conjunctiva 13, iris 14, optic nerve 15,sclera 16, macula lutea or macula 17, lens 18, retina 20, choroid 22,and fovea 41 are indicated. The macula is located in the center of theposterior part of the retina 20 and is the most sensitive portion of theretina. It is an oval region of about 3 mm by 5 mm, in the center ofwhich is a depression, the fovea centralis 41, from which rods areabsent. Inside the fovea 41 is the point of entrance of the optic nerve15 and its central artery. At this point; the retina 20 is incompleteand forms the blind spot.

The encircled area 2 of FIG. 1 is shown in exploded form in FIG. 2. Asshown in FIG. 2, the retina 20 forms the innermost layer of theposterior portion of the eye and is the photoreceptor organ. The retina20 has an optical portion that lines the inner surface of the choroid 22and extends from the papilla of the optic nerve 15 to the ora serrata 21anteriorly. At the papilla, where the retina 20 stops, and at the oraserrata 21, the retina 20 is firmly connected with the retinal pigmentepithelium (RPE) 101.

The retina 20 has ten parallel layers. These are, from the choroid in,as follows: the RPE 101, photoreceptor cells (rod and cone inner andouter segments) 102, the external limiting membrane 103, the outernuclear layer 104, the outer plexiform layer 105, the inner nuclearlayer 106, the inner plexiform layer 107, the layer of ganglion cells108, the layer of optic nerve fibers or neurofiber layer 109, and theinternal limiting membrane 110. The internal limiting membrane 110 isvery thin (less than 5 μm), and normally adheres with the neurofiberlayer 109 of the ganglion cells 108.

The pigment epithelial cell layer or RPE 101 rests on a basal laminatermed Bruch's membrane 112 that is adjacent to the choroid 22.

The next three layers are composed of various portions of one cell type,termed the first neuron. These layers are the photoreceptor region(lamina) 102 of rods and cones, the external limiting membrane 103, andthe outer nuclear layer 104 composed of the nuclei of the rods and conescells. The rods have long, thin bodies, and the cones have a broad base.The rods have greater sensitivity for low light levels; the cones havebetter visual acuity in daylight and are also responsible for colorperception. There are three types of cones, each absorbing light from adifferent portion of the visible spectrum: long-wavelength (red),mid-wavelength (green), and short-wavelength (blue) light. Both rods andcones contain the transmembrane protein opsin, and the prosthetic groupretinal, a vitamin A derivative. The opsins in each cell type containdifferent amino acids that confer differences in light absorption.

The RPE, photoreceptor cells, external limiting membrane, outer nuclearlayer, and outer plexiform layer constitute the neuro-epithelial layerof the retina.

The inner nuclear layer, inner plexiform layer, ganglion cell layer,nerve fiber layer, and internal limiting membrane constitute thecerebral layer of the retina. The inner nuclear layer contains bipolarcells, ganglion cells, horizontal cells, amacrine cells, Muller cells,and astrocytes, the latter two being types of glial cells. The Mullercells have nuclei in the inner nuclear area and cytoplasm extending fromthe internal limiting membrane 110 to the external limiting membrane103. The external limiting membrane 103 is a region of terminal barsbetween Muller's cells and the visual receptors.

The next three layers of the retina are composed of various parts of thesecond neurons, whose nuclei reside in the inner nuclear layer and whosecytoplasmic processes extend into the outer plexiform layer to synapsewith the receptor cells and to the inner plexiform layer to synapse withthe ganglion cells. Thus, the second neuron is bipolar.

The third neuron, the multipolar ganglion cells, sends its nerve fiber(axon) to the optic nerve.

The last layer of the retina is the internal limiting membrane (ILM) onwhich the processes of the Muller's cells rest.

The retina contains a complex interneuronal array. Bipolar cells andganglion cells are sensory cells that together form a path from the rodsand cones to the brain. Other neurons form synapses with the bipolarcells and ganglion cells and modify their activity. For example,ganglion cells, or ganglia, generate action potentials and conduct theseimpulses back to the brain along the optic nerve. Vision is based on themodulation of these impulses, but does not require the directrelationship between a visual stimulus and an action potential. Thevisual photosensitive cells, the rods and cones, do not generate actionpotentials, as do other sensory cells (e.g., olfactory, gustatory, andauditory sensory cells).

Muller cells, the principal type of glial cells, form architecturalsupport structures stretching radially across the thickness of theretina, and forming the limits of the retina at the outer and innerlimiting membranes, respectively. Muller cell bodies in the innernuclear layer project irregularly thick and thin processes in eitherdirection to the outer and inner limiting membranes. These processesinsinuate themselves between cell bodies of the neurons in the nuclearlayers, and envelope groups of neural processes in the plexiform layers.Retinal neural processes can only have direct contact, withoutenveloping Muller cell processes, at their synapses. The junctionsforming the outer limiting membrane are between Muller cells, and otherMuller cells and photoreceptor cells, as sturdy desmosomes or zonulaadherens. Muller cells perform a range of functions that contribute tothe health of the retinal neurons. These functions include supplyingendproducts of anaerobic metabolism (breakdown of glycogen) to fuelneuronal aerobic metabolism; removing neural waste products such ascarbon dioxide and ammonia and recycling spent amino acid transmitters;protecting neurons from exposure to excess neurotransmitters usinguptake and recycling mechanisms; phagocytosis of neuronal debris andrelease of neuroactive substances; synthesizing retinoic acid, requiredin the development of the eye and nervous system, from retinol;controlling homeostasis and protecting neurons from deleterious changesin their ionic environment by taking up and redistributing extracellularK⁺; and contributing to generation of the electroretinogram (ERG)b-wave, the slow P3 component of the ERG, and the scotopic thresholdresponse (STR) by regulating K⁺ distribution across the retinal vitreousborder, across the whole retina, and locally in the inner plexiformlayer of the retina.

Astrocytes, the other type of glial cell, envelope ganglion cell axonsand have a relationship to blood vessels of the nerve fiber, suggestingthey are axonal and vascular glial sheaths and part of a blood-brainbarrier. They contain abundant glycogen, similar to Muller cells, andprovide nutrition to the neurons in the form of glucose. They may servea role in ionic homeostasis in regulating extracellular K⁺ levels andneurotransmitter metabolism. They have a characteristic flattened cellbody and fibrous radiating processes which contain intermediatefilaments. The cell bodies and processes are almost entirely restrictedto the nerve fiber layer of the retina. Their morphology changes fromthe optic nerve head to the periphery: from extremely elongated near theoptic nerve to a symmetrical stellate form in the far peripheral retina.

Microglial cells are not neuroglial cells and enter the retinacoincident with mesenchymal precursors of retinal blood vessels indevelopment, and are found in every layer of the retina. They are one oftwo types. One type is thought to enter the retina at earlier stages ofdevelopment from the optic nerve mesenchyme and lie dormant in theretinal layers for much of the life of the retina. The other typeappears to be blood-borne cells, possibly originating from vesselpericytes. Both types can be stimulated into a macrophagic function uponretinal trauma, in degenerative diseases of the retina, etc. when theythen engage in phagocytosis of degenerating retinal neurons.

All glial cells in the central nervous system (CNS) are coupledextensively by gap junctions. This coupling underlies several glial cellprocesses, including regulating extracellular K⁺ by spatial buffering,propagating intercellular Ca²⁺ waves, regulating intracellular ionlevels, and modulating neuronal activity.

Activation of retinal glial cells with chemical, mechanical, orelectrical stimuli often initiate propagated waves of calcium ions(Ca²⁺). These Ca²⁺ waves travel at a velocity of 23 μm/second and up to180 μm/second from the site of initiation. The waves travel through bothastrocytes and Muller cells, even when the wave is initiated bystimulating a single astrocyte.

Ca²⁺ waves propagate between glial cells in the retina by twomechanisms: diffusion of an intracellular messenger through gapjunctions, and release of an extracellular messenger. Ca²⁺ wavepropagation between astrocytes is mediated largely by diffusion of anintracellular messenger, likely inositol triphosphate (IP3), through gapjunctions, along with release of adenosine triphosphate (ATP).Propagation from astrocytes to Muller cells, and from one Muller cell toother Muller cells, is mediated by ATP release.

Retinal neurons and glial cells also communicate. Muller cells havetransient Ca²⁺ increases that occur at a low frequency. Stimulating theretina with repetitive light flashes significantly increases thefrequency of these Ca²⁺ transients, most prominent in Muller cellendfeet at the retinal surface, but also in Muller cell processes in theinner plexiform layer. This neuron-to-glial cell communication indicatesthat glial cell Ca²⁺ transients are physiological responses in vivo.

Stimulated glial cells directly modulate the electrical activity ofretinal neurons, leading either to enhanced or depressed neuronalspiking. Inhibitory glial modulation of neuronal spiking may beCa²⁺-dependent, because the magnitude of neuronal modulation wasproportional to the amplitude of the Ca²⁺ increase in neighboring glialcells. Glial cells can modulate neuronal activity in the retina by atleast three mechanisms. In some ganglion cells, glial cell activationfacilitates synaptic transmissions and enhances light-evoked spiking. Inother ganglion cells, there is depressed synaptic transmissions anddecreased spiking. Glial cell activation can also result in ganglioncells hyperpolarization, mediated by activating A1 receptors and openingneuronal K⁺ channels.

Stimulated glial cells also indirectly modulate the electrical activityof retinal neurons. This is mediated by glutamate uptake by Muller cellsat synapses by glutamate transporters such as GLAST (EAAT1) and GLT-1(EAAT2) in Muller cells. When glutamate transport in the retina isblocked, both the amplitude and the duration of ganglion cell EPSCs areincreased. Glial cell modulation of electrical activation of retinalneurons is also mediated by regulating extracellular K⁺ and H⁺ levels.Neuronal activity leads to substantial variations in the concentrationof K⁺ and H⁺ in the extracellular space, which can alter synaptictransmission; an increase of K⁺ depolarizes synaptic terminals, while anincrease of H⁺ blocks presynaptic Ca²⁺ channels and NMDA receptors.Muller cells regulate extracellular concentrations of K⁺ and H⁺, thusinfluencing the effect of these ions on synaptic transmission.

With reference to FIG. 2, one skilled in the art will appreciate thatsolar cell micro- and/or nano-particles 125, provided selectively orsubstantially throughout the all regions of the retina, enhance,facilitate or boost the ability of these biological cells to regulatetheir polarity. This is in contrast to use of a device that supplies anelectrical potential, that is implanted in an invasive surgicalprocedure, that is localized, etc.

Besides pathologies in one or more of the above described mechanisms tomaintain and/or regulate retinal cell polarity, other excitable cellsbesides the retina may have pathologies that occur from defects in cellplasma membrane polarization. As one example, excitable cells in thebrain of Alzheimer's patients have abnormal electrical conducting andstabilizing mechanisms, resulting in loss of electrical stimulation.Repolarization of these cells provides additional stimulation to replacethe abnormal cell membrane polarization and/or the cell membranepolarization that was diminished or lost. As another example, glial cellscar tissue culminating from epileptic seizures results in abnormalelectrical stabilizing mechanisms in excitable cells of the brain.Repolarization of these cells provides a stabilized threshold, resultingin a calming effect. One skilled in the art will appreciate otherpathologies for which the inventive method may be used.

The inventive method includes mechanisms to delay, minimize, reduce,alleviate, correct, or prevent electro-sensory polarization pathologies.Such mechanisms may attenuate cellular damage resulting from abnormalpolarization, reduced polarization, enhanced polarization,hyperpolarization, or loss of polarization. These polarization defectsmay be of any type and/or cell combination, and may stimulate and/orde-stimulate the cell(s). They may, for example, be transient in onecell type, sustained in one cell type, propagated to affect adjacentcells, propagated along a network to affect non-adjacent cells, etc.

It is known attaching nanocrystal quantum dots to semiconductor layersincreases the photovoltaic efficiencies. The semiconductor solar cellswork by using the energy of incoming photons to raise electrons from thesemiconductor's valence band to its conduction band. A potential barrierformed at the junction between p-type and n-type regions of thesemiconductor forces the pairs to split, thereby producing a current,thus influencing, changing, or regulating the polarization of amembrane. The particles are stimulated by using an external or internalenergy source. Polarization of the particles is regulated by producingor varying the current. The particles are used to stimulate the cellmembrane by varying the input energy from the energy source.

One embodiment provides nano- or micro-sized solar cells to regulate thepolarity of excitable cells. As previously described, excitable cellsinclude, but are not limited to, sensory cells such as the retina of theeye, all three types of muscle cells, and central and peripheral systemnerve cells. Such nano- or micro-sized solar cells are hereinaftergenerally referred to as particles 125 as shown in FIG. 2. Particlesencompass any and all sizes which permit passage through intercellularand/or intracellular spaces in the organ or area of the organ ofinterest. For example, intercellular spaces in the retina are about 30angstroms (30×10⁻⁸), so that particles for intercellular retinaldistribution may be sized for these spaces, as known to one skilled inthe art.

The solar cell nano- and/or micro-particles 125 are three dimensionalsemiconductor devices. The particles use light energy or ultrasoundenergy to generate electrical energy to provide a photovoltaic effect.In one embodiment, the particle material is a ceramic. In anotherembodiment, the particle material is a plastic. In another embodiment,the particle material is silicon. Particles of crystalline silicon maybe monocrystalline cells, poly or multicrystalline cells, or ribbonsilicon having a multicrystalline structure. These are fabricated asmicroscale or nanoscale particles that are administered to a patient.

The particles may be a nanocrystal of synthetic silicon,gallium/arsenide, cadmium/selenium, copper/indium/gallium/selenide, zincsulfide, indium/gallium/phosphide, gallium arsenide, indium/galliumnitride, and are synthesized controlling crystal conformations andsizes.

The particles (quantum dots) may also be biocompatible short peptidesmade of naturally occurring amino acids that have the optical andelectronic properties of semiconductor nano-crystals. One example isshort peptides of phenylalanine. The particles can consist of bothinorganic or organic materials, as previously described.

The particles may be coated with biocompatible mono- or bilayers ofphospholipid a protein, a peptide polyethylene glycol (PEG) that can beused as a scaffold to aid in biocompatibility of the particle. Theparticles can be entirely or partially biodegradable.

In one embodiment, the quantum dots are delivered to the retinal cellcytoplasm or nucleus, regardless of the particular injection site in theeye. In one embodiment, the quantum dots are introduced into the centralnervous system, e.g., by injection. In one embodiment, the quantum dotsare covalently linked, i.e., conjugated, with natural or syntheticbiomolecules (e.g., proteins, peptides, nucleic acids, oligonucleotides,etc.) that introduce a vector (e.g., adeno-associated virus (AAV) forretinal gene therapy. Such a vector and/or quantum dots can be labeledfor visualization, tracking, sensing, etc. For example, the quantum dotscan be labeled or tagged with a signal recognition moiety. Such a vectorcan incorporate quantum dots into the viral capsid using, e.g.,(poly)ethylene glycol (PEG) moieties. Combinations of these embodimentsare contemplated and included in the inventive method, using methodsknown by one skilled in the art and as subsequently described.

In one embodiment, quantum dots are conjugated with a moiety such as anocular peptide or protein, to result in a biologically active quantumdot conjugate. Such conjugation allows the therapeutic effect to becontrolled and specific, while sensing and tracking the conjugatelocation, function, etc. in, e.g., the retina.

Examples of such ocular peptides and proteins include, but are notlimited to, membrane-bound G-protein coupled photoreceptors (opsins,including the rod cell night vision pigment rhodopsin and cone cellcolor vision proteins), and members of the family of ocular transportproteins (aquaporins).

In one embodiment, short peptides of naturally occurring amino acidsthat have the optical and electronic properties of semiconductornano-crystals are conjugated to quantum dots. One non-limiting exampleof such a short peptide is (poly)phenylalanine. In these embodiments,the resulting conjugate contains both inorganic and organic materials,as previously described. In one embodiment, the conjugates may be coatedwith biocompatible mono- or bilayers of phospholipid, protein, and/or a(poly)ethylene glycol (PEG) molecule that can be used as a scaffold toaid in biocompatibility of the particle. Any of these organic moietiesmay be utilized to ionically, electronically or covalently form thebiologically active conjugates. The conjugates are entirely or partiallybiodegradable.

In one embodiment, a quantum dot conjugated to a vector is capable ofmodifying an ocular gene, e.g., a gene of a retinal cell. In thisembodiment, the quantum dot, besides regulating membrane polarity of anexcitable cell such as a retinal cell, also provides therapy toameliorate or prevent a genetically based retinal disease (e.g.,retinitis pigmentosa). In one embodiment, the vector may be a plasmidvector, a binary vector, a cloning vector, an expression vector, ashuttle vector, or a viral vector as known to one skilled in the art.The vector typically contains a promoter, a means for replicating thevector, a coding region, and an efficiency increasing region. In oneembodiment, the vector is a virus such as an adenovirus, anadeno-associated virus (AAV), a retrovirus, and other viral vectors forgene therapy, as known to one skilled in the art. As one non-limitingexample, quantum dots are functionalized and/or linked to viral vectorsusing (poly)ethylene glycol (PEG) moieties. The number of PEGS can bevaried depending on, e.g., ocular site, need to enhanced hydrophilicity,protein size, etc. The viral vector and quantum dot are combined in thepresence of at least one biocompatible adjuvant, suspension agent,surfactant, etc. Quantum dots may be coated with or linked to, e.g.,folate, polydopamine, etc. so that these molecules are targetedintracellularly, extracelluarly, to a cell membrane, to a specificcellular site or organelle, etc.

Conjugation of quantum dots to viral capsids permits in vivo observationof retinal neurons and the individual glycine receptors in livingneurons. A single quantum dot can be recognized by optical coherencetomography (OCT) and can be counted, tracked, assessed, monitored, andevaluated for longevity and efficacy, and hence therapy can also bemonitored, over time.

In one embodiment, quantum dots associated with other biomolecules,e.g., conjugated with halorrhodopson, conjugated with a customizedvirus, are used to regulate, i.e., stimulate or inhibit, actionpotential of a neuron. Quantum dots can be associated with, e.g.,conjugated with, a virus, a virus capsid, a cell penetrating protein,and/or other molecule(s) to stimulate specific neurons or specificneuronal function, or may be provided with appropriate stem cells.

In one embodiment, covalent conjugation may not be required or desired,and in this embodiment quantum dots may be simply associated with aviral vector. In one embodiment, quantum dots may be mixed with anappropriate viral vector in the presence of a cationic polymer, e.g.hexadimethrine bromide POLYBRENE® to form a colloidal complex suitablefor introducing into a retinal cell. In one embodiment, quantum dots aretagged with an amide, a thiol, etc. using electrostatic interactionalong with functionalizing means known to one skilled in the art.

In some embodiments, it may be useful to assess, monitor, track,evaluate location, evaluate stability, etc. of the quantum dotsconjugated or otherwise associated with a moiety as previouslydescribed. In these embodiments, the quantum dots are tagged with arecognition moiety to provide a signal, and may themselves be conjugatedto another biologically active moiety, e.g., DNA, RNA, peptide, protein,antibody, enzyme, receptor, etc., as known to one skilled in the art.Tagging may be effected via a covalent bond with a amide, thiol,hydroxyl, carbonyl, sulfo, or other such group on the biologicallyactive moiety, as well known to one skilled in the art.

While each solar cell particle is oriented, the plurality of particlesprovided in the body are not uniformly directionally oriented, nor dothey require a backing layer to maintain orientation or position. Theyhave a positive-negative (P-N) junction diode and may be constructed aseither negative-intrinsic-positive (NIP) or positive-intrinsic-negative(PIN), as known to one skilled in the art.

As an example, p-type silicon wafers, and doped p-type silicon wafers toform n-type silicon wafers, are contacted to form a p-n junction.Electron diffuse from the region of high electron concentration, then-type side of the junction, into the region of low electronconcentration, the p-type side of the junction. When the electronsdiffuse across the p-n junction, they recombine with an electrondeficiency (holes) on the p-type side. This diffusion of carriers doesnot happen indefinitely however, because of the electric field createdby the imbalance of charge immediately either side of the junction whichthis diffusion creates. Electrons from donor atoms on the n-type side ofthe junction cross into the p-type side, leaving behind the (extra)positively charged nuclei of the group 15 (V) donor atoms such asphosphorous or arsenic, leaving an excess of positive charge on then-type side of the junction. At the same time, these electrons arefilling holes on the p-type side of the junction and are becominginvolved in covalent bonds around the group 13 (III) acceptor atoms suchas aluminum or gallium, making an excess of negative charge on thep-type side of the junction. This imbalance of charge across the p-njunction sets up an electric field which opposes further diffusion ofcharge carriers across the junction. The region where electrons havediffused across the junction is called the depletion region or the spacecharge region because it no longer contains any mobile charge carriers.The electric field which is set up across the p-n junction creates adiode, allowing current to flow in only one direction across thejunction. Electrons may pass from the n-type side into the p-type side,and holes may pass from the p-type side to the n-type side. Because thesign of the charge on electrons and holes is opposite, current flows inonly one direction. Once the electron-hole pair has been created by theabsorption of a photon, the electron and hole are both free to move offindependently within a silicon lattice. If they are created within aminority carrier diffusion length of the junction, then, depending onwhich side of the junction the electron-hole pair is created, theelectric field at the junction will either sweep the electron to then-type side, or the hole to the p-type side.

One embodiment of the invention uses nanocrystals of semiconductormaterial referred to as quantum dots (Evident Technologies, Troy N. Y.;Oceano NanoTech, Springdale Ak.). Nanocrystal solar cells are solarcells based on a substrate with a coating of nanocrystal. Thenanocrystals are typically based on silicon, CdTe or CIGS and thesubstrates are generally silicon or various organic conductors. Quantumdot solar cells are a variant of this approach. These have a compositionand size that provides quantum properties between that of singlemolecules and bulk materials, and are tunable to absorb light over thespectrum from visible to infrared energies. Their dimensions aremeasured in nanometers, e.g., diameter between about 1 nm to about 100nm. When combined with organic semiconductors selected to have thedesired activation properties, they result in particles with selectablefeatures. The particles can also have passive iron oxide coatings withor without polyethylene glycol coatings or positive charge coatings ascommercially provided. Quantum dot solar cells take advantage of quantummechanical effects to extract further performance.

Nanocrystals are semiconductors with tunable bandgaps. The quantum dotnanocrystal absorption spectrum appears as a series of overlapping peaksthat get larger at shorter wavelengths. Because of their discreteelectron energy levels, each peak corresponds to an energy transitionbetween discrete electron-hole (exciton) energy levels. The quantum dotsdo not absorb light that has a wavelength longer than that of the firstexciton peak, also referred to as the absorption onset. Like otheroptical and electronic properties, the wavelength of the first excitonpeak, and all subsequent peaks, is a function of the composition andsize of the quantum dot. Smaller dots result in a first exciton peak atshorter wavelengths.

The quantum dots may be provided as a core, with a shell or coating ofone or more atomic layers of an inorganic wide band semiconductor. Thisincreases quantum yield and reduces nonradiative recombination,resulting in brighter emission provided that the shell is of a differentsemiconductor material with a wider bandgap than the core semiconductormaterial. The higher quantum yield is due to changes in the surfacechemistry of the core quantum dot. The surface of nanocrystals that lacka shell has both free (unbonded) electrons, in addition to crystaldefects. Both of these characteristics tend to reduce quantum yield bypermitting nonradiative electron energy transitions at the surface. Ashell reduces opportunities for nonradiative transitions by givingconduction band electrons an increased probability of directly relaxingto the valence band. The shell also neutralizes the effects of manytypes of surface defects.

The quantum dots may respond to various wave lengths of electromagneticradiation, i.e., visible, invisible, ultrasound, microwaves. The quantumdots respond by emitting an electrical potential or fluoresce whenexposed to electromagnetic radiation. The quantum dots may be made, orself-assembled, from CdSe and a shell of zinc gallium arsenide, indiumgallium selenide, or cadmium telluride. Luminescent semiconductorquantum dots such as zinc sulfide-capped cadmium selenide may becovalently coupled to biomolecules for use in ultrasensitive biologicaldetection. These nanometer-sized conjugates are water-soluble andbiocompatible.

Quantum dots, organic quantum dots or solar cells, may be made from allorganic molecules such as organic nanocrystal solar cells, polymers,fullerenes, etc. Quantum dots may be coated with organic molecules,biocompatible proteins, peptides, phospholipids, or biotargetedmolecules etc., or covalently attached to polyethylene glycol polymers(i.e., they may be PEGylated) to last longer. These quantum dots, ordevices containing quantum acts are amenable to large scale production.They may be built from thin films, polymers of organic semiconductors.These devices differ from inorganic semiconductor solar cells in thatthey do not rely on the large built-in electric field of a PN junctionto separate the electrons and holes created when photons are absorbed.The active region of an organic device consists of two materials, onewhich acts as an electron donor and the other as an acceptor. The shortexcitation diffusion lengths of most polymer systems tend to limit theefficiency of such devices. However, quantum dots can be used for cellmembrane stimulation.

The quantum dots can be made to respond to various wavelengths of light(visible and invisible). In one embodiment they are coated with organicmolecules. In one embodiment, they are completely organic. In oneembodiment, they are PEGylated to last longer. In one embodiment, theyare coated to be attracted to certain receptors or stay only on the cellsurface.

Bioelectrical signals exist in all cells and play an important role inallowing the cells to communicate with each other. Quantum dots canfacilitate these signal transmission between the cells, such as throughcell membranes and their membrane potentials, thereby maintaining normalfunction in the tissue which include cell survival and growth,individually or collectively. Quantum dots can enhance regeneration ofthe cells. Quantum dots can enhance neural axons and enhance the woundhealing process.

Cell activity relates to depolarization and re-polarization of the cellmembrane. Quantum dots can regulate polarization and depolarization andthus enhance the action potential of the membrane. Lack of cell activityleads to cell atrophy. Similarly, loss of the cell membrane potentialcauses cell degeneration and atrophy. The therapeutic effects of quantumdots are achieved by the effects that quantum dots exert on membranepotential when stimulated, e.g. light, photoelectrical, ultrasound, etc.In the eye and in the nervous system, quantum dots can be stimulated(e.g., through the cornea, sclera or skull etc. for the brain, spinalcord, and nerves), thus enhancing or maintaining the cell membranepotential (e.g., nerve cell, glial cells, astrocytes, etc.). Thisprocess preserves the function of such cells (nerve cells, glial cells,astrocytes, etc.) by maintaining their membrane potentials, thusmaintaining cell viability and function.

In one embodiment, the method and concept is applied to the eye. In oneembodiment, the method and concept is applied to the brain and spinalcord nerve cells and axons. In this embodiment, the method is used toenhance or stimulate regrowth of nerve cells, axons, and/or other brainand spinal cord tissue.

In one embodiment, the effects of quantum dots on the cells can beenhanced by combining quantum dots with growth factors. Such growthfactors are known to one skilled in the art, and include but are notlimited to nerve growth factors, glial growth factors, placenta growthfactor, etc. In one embodiment the effects of quantum dots on the cellscan be enhanced by administering and/or regulating quantum dotsessentially simultaneously with certain pharmaceuticals or agents,including but not limited to TAXOL®, carbonic anhydrase inhibitors, etc.Quantum dots, when activated by light, enhance drug penetration throughthe cell membrane. This can be used therapeutically in combination withmany medications which may not penetrate the cell membrane easilybecause of their chemical structures. However, this concept can be usedalso in conjunction with antibiotics, antifungal agents, etc. to killthe organism that caused skin or mucosa ulcers resisting therapy.

The treatment can be done easily by topically applying quantum dotsalong with the appropriate medication and using light to activate thequantum dots. The method of delivery to the eye may be by injection, eyedrops, ointments, sprays or other applications to treat an optic nerve.The method of delivery to the brain may be by injection of the quantumdots into cerebrospinal fluid, brain ventricles, intra-ocularly, oradministration by nasal sprays or drops. The method of delivery to theskin or mucosa, e.g., nasal mucosa, is by spraying. Most of theseapplications avoid possible systemic side effects. The size of theparticles allows them to easily diffuse into tissues. For neuralapplications other than the eye, quantum dots, either conjugated orassociated with a drug, and/or administered without a drug or otheragent, are administered by any route of delivery including but notlimited to local, systemic, injection in the CNS, by nasal routes, e.g.,spray, drops, to regulate the nasal olfactory nerve, or localizedinjection in the vicinity of the peripheral nerves or ganglions, etc.

In one embodiment, the inventive method is used in a patient with aneurological disorder. While described in detail for use in a patientwith epilepsy, which is a common neurological disorder requiringtreatment, the inventive method is not so limited and encompasses anyneurological disorder of the central and/or peripheral nervous system.Epilepsy is thus used an exemplary but non-limiting embodiment of use ofthe method.

Epilepsy is a chronic condition that transiently affects about 50million individuals. It is not a single disorder, but instead is a groupof syndromes with vastly divergent symptoms. Its unifying and diagnosticfeature is episodic abnormal electrical activity in the brain thatresults in seizures. These seizures are transient, recurrent, andunprovoked; signs and/or symptoms of abnormal, excessive, or synchronousneuronal activity in the brain. All seizures involve loss ofconsciousness; types of seizures are characterized according to theireffect on the body. These include absence (petit mal), myoclonic,clonic, tonic, tonic-clonic (grand mal), and atonic seizures.

Some forms of epilepsy are confined to particular stages of childhood.In children, epilepsy may result from genetic, congenital, and/ordevelopmental abnormalities. In adults over 40, it may result fromtumors. At any age, it may result from head trauma and central nervoussystem infections. Post-traumatic epilepsy (PTE) is a form of epilepsythat results from brain damage caused by physical trauma to the brain:traumatic brain injury (TBI). An individual with PTE suffers repeatedpost-traumatic seizures (PTS) more than a week after the initial injury.PTE can also occur after infectious diseases involving the CNS orperipheral nerves.

Epilepsy is usually controlled, but not cured, with medication, althoughsurgery is sometimes needed. Therapeutic agents include (a)sodium-channel blockers (voltage dependent). (b) calcium channelblockers (T-type), (c) potentiators of GABA (inhibitory), and (d) thosethat decrease excitatory transmission (glutaminic).

Some medication, administered daily, may prevent seizures altogether orreduce their frequency. Such medications, termed anticonvulsant drugs orantiepileptic drugs (AEDs), included valproate semisodium (Depakote,Epival), valproic acid (Depakene, Convulex), vigabatrin (Sabril), andzonisamide (Zonegran). A problem is that all have idiosyncratic andnon-dose-dependent side effects. Thus, one cannot predict which patientson a therapeutic regimen will exhibit side effects or at what dose.

Some medications are commonly used to abort an active seizure or tointerrupt a seizure flurry. These include diazepam (Valium) andlorazepam (Ativan). Drugs used only in the treatment of refractorystatus epilepticus include paraldehyde (Paral), midazolam (Versed), andpentobarbital (Nembutal).

Bromides, the first of the effective anticonvulsant pure compounds, areno longer used in humans due to their toxicity and low efficacy.

Palliative surgery for epilepsy is intended to reduce seizure frequencyor severity. For example, a callosotomy or commissurotomy is performedto prevent seizures from generalizing, i.e., from being transmitted tothe entire brain, which results in loss of consciousness

Vagus nerve stimulation (VNS) controls seizures with an implantedelectrical device, similar in size, shape, and implant location to apacemaker. The implanted VNS device connects to the vagus nerve in theneck and is set to emit electronic pulses to stimulate the vagus nerveat pre-set intervals and milliamp levels. About 50% of individuals withan implanted VNS device showed significantly reduced seizure frequency.

The Responsive Neurostimulator System (tRNS), in clinical study prior toregulatory approval, is a device implanted under the scalp with leadsimplanted either on the brain surface or in the brain close to the areawhere the seizures are believed to start. At the outset of a seizure,small amounts of electrical stimulation are delivered to the brain tosuppress the seizure. The RNS system differs from the VNS: the RNSsystem is patient responsive in that it directly stimulates the brain,whereas the VNS system provides physician-determined pre-set pulses atpredetermined intervals. The RNS system is designed to respond todetected signs that a seizure is about to begin and can record eventsand allow customized response patterns that may provide a greater degreeof seizure control.

One class of therapeutic agents for treating epilepsy are the carbonicanhydrase inhibitors, but all have undesirable side effects.

Acetazolamide (Acz), a known inhibitor of carbonic anhydrase, is onesuch agent. It prevents hypoxic pulmonary vasoconstriction (HPV) andthus is also used to treat altitude, sickness, glaucoma, and benignintracranial hypertension. Acetazolamide, however, affects kidneyfunction because it reduces NaCl and bicarbonate reabsorption in thekidney proximal tubule. The reduction results in a mild diuretic effect,although it is partially compensated by the kidney distal segment andthe metabolic acidosis produced by the bicarbonaturia. Methazolamide,also a carbonic anhydrase inhibitor, is longer-acting than acetazolamidewith fewer kidney effects. Dorzolamide, a sulfonamide and topicalcarbonic anhydrase II inhibitor, reduces the elevated intraocularpressure in patients with open-angle glaucoma or ocular hypertensionthat are insufficiently responsive to beta-blockers. Inhibition ofcarbonic anhydrase II in the ciliary processes of the eye decreasesaqueous humor secretion, presumably by slowing the formation ofbicarbonate ions with subsequent reduction in sodium and fluidtransport. Topiramate is a weak inhibitor of carbonic anhydrase,particularly subtypes II and IV. It is a sulfamate-substitutedmonosaccharide that is related to fructose. In is approved in the U.S.as an anticonvulsant to treat epilepsy, migraine headaches, andLennox-Gastaut syndrome. Its inhibition of carbonic anhydrase may besufficiently strong to result in clinically significant metabolicacidosis.

Acetazolamide and other calcium-inhibiting sulfonamides increaseintracellular pH and relax mesenteric arteries preconstricted withnorepinephrine. Calcium inhibitors and/or the intracellularalkalinization activate a calcium-dependent potassium channel, resultingin hyperpolarization of the vascular smooth muscle cell, reduction ofvoltage-dependent calcium channel activity, decreased intracellularcalcium, and vasorelaxation.

Spreading depression (SD) is a pathophysiologic event characterized bydepressed EEG activity and a change of the direct current potential asan indicator of a short-lasting cell membrane depolarization. It may beinduced by a variety of cortical stimuli, including potassium chlorideor glutamate application, and electrical or mechanical stimulation; italso occurs secondary to ischemia. It is accompanied by severe changesin ion homeostasis and water shifts from the extracellular tointracellular space, mirrored by changes of electrical impedance anddirect current (DC) potential. The area of depolarization spreads alongcortical tissue like a wave, moving away from the initiation site towardthe periphery, and propagates with an estimated velocity of 3 mm/min.Electrical measurements from the cortex surface show negative deflectionof the DC potential, lasting 1 to 2 minutes, combined with EEGsuppression. Under normoxic conditions, SD is not followed by permanentneuronal damage, and the depressed neuronal activity is compensated byincreased glucose metabolism and blood flow during the repolarizationphase. The cell membrane repolarization requires an enormous metaboliceffort and is compensated by increased glucose metabolism and increasedblood flow.

Serotonin homeostasis, regulated by serotonin receptor 1A (Htr1a), isrequired for normal serotonin levels. Htr1a also mediates autoinhibitionof serotonin production; excessive serotonin autoinhibition isassociated with sporadic autonomic dysregulation and death. Tryptophan,a serotonin precursor, increases serotonin production. Administration ofthe selective Htr Ia antagonist WAY100635 completely shuts downserotonin-induced neuron impulses, resulting in apnea preceded bybradycardia; both lung function and heart function were affected.

Spreading depression (SD) has been extensively studied and is likely animportant mechanism in several human diseases. Cerebral hemodynamics,i.e., cerebral blood volume and water changes, were assessed byhigh-speed MRI during potassium-induced spreading depression. MRIimages, and brain voltage readings, were used to determine apparentdiffusion coefficients over time that correlated with potassium fluxalong the cortex. Acetazolamide treatment resulted in vasodilation andarrested spreading depression.

Diffusion-weighted imaging is highly sensitive to slowing water protontranslations early in the ischemic episode, i.e., within minutes. MRimaging measured the ADC of brain water decreases by 30% to 60%, andrecent findings suggested significant apparent diffusion slowing (ADCdecreases) in stroke results predominantly due to cellular swelling andreflects a shift of relatively faster translating extracellular waterprotons into a more hindered intracellular environment. It has beenshown that when the Na⁺/K⁺ pump is disabled by intraparenchymal ouabain,the ADC decreases. This supports a link between altered ion homeostasisand alteration in ADC. There is a relation between membrane polarizationand diffusion as measured by the ADC. Failure of the transmembrane ionpumps and subsequent loss in cell membrane potential is immediatelyfollowed by disruption of ion homeostasis. The resulting ionic imbalancecauses an osmotically driven flow of water into the cells. MR imagingindicates the subsequent cell swelling with restricted extracellular orintracellular diffusion, and increased extracellular tortuosity, reducesthe ADC.

The concept of cell preservation by quantum dots administration andtreatment applies to the above these diseases and reduces degenerationof all brain cells (nerve cells, glial cells, etc.).

Quantum dots are useful in providing repeated electric pulses either tothe brain, spinal cord, or isolated nerve cells that are involved withvarious neural disorders. In disorders involving these regions low levelbrain, spinal cord, etc. neural pulses are not passing through for onereason or another, e.g., synapses, scar, misdirection, etc., and arereleased either as a giant pulse or can circuit back and forth until themembrane potential is completely exhausted. Therefore a pulsedstimulation by an external source, such as light or electric pulsesapplied to the brain, ventricles, spinal cord, cerebrospinal fluid,having quantum dots would eliminate an avalanche of the pulses inposttraumatic epilepsy, restless leg syndrome, spinal cord epilepsy,etc. A version of this concept could be potentially used to modify brainwaves needed for sound sleep, alleviation of depression, etc.Stimulation of the olfactory nerve can enhance neuronal regeneration inthe brain in aging adults or in Alzheimer's disease or slow itsprogression.

A physician may select specific properties and emission frequencies toselectively regulate polarization in specific sites and for specificresults. Thus, the particles are tunable to provide desired properties;for example, they may be size specific, current specific, patientspecific, disease specific, activation specific, site specific, etc.

As one example, particles provided throughout the retinal layers may beselectively regulated to normalize polarization and/or reduce or preventrepolarization, depolarization, and/or hyperpolarization. As anotherexample, selected particles may be administered to selected sites andselectively regulated (e.g., temporally, spatially, activationally, etc)to result in different effects to fine-tune a desired outcome. Morespecifically, a patient's progress may be monitored after a slightregulation and, if warranted, further regulation may be administereduntil a desired outcome is obtained. For example, a patient with muscletremors may be treated with the inventive method for a duration, extent,activation energy, etc. to selectively repolarize striated muscle cellsuntil a desired effect is reached.

In one embodiment, the particles are mixed into or with a biocompatiblefluid. In another embodiment, the particles are in the form of beads orspheres. In another embodiment, the particles are provided as a film. Inanother embodiment, the particles are drawn and provided as fibers. Inany of these embodiments, the particles are provided to a patient byinjection to other minimally invasive techniques known to one skilled inthe art.

Upon administration, the particles are disseminated and/or locatedintracellularly (within a cell), intercellularly (between cells), orboth intracellularly and intercellularly. They may be administered in anumber of ways. With respect to the eye, they may be injected throughthe retina, under the retina superiorly, over the fovea, through theouter plexiform layer down to the fovea, into the vitreous cavity todiffuse through the retina, etc. The procedure permits particles to belocated at any site including the macula, that is, the particles may bedirectly on the macula; directly on the fovea, etc. distinguishing fromprocedures requiring electrodes to be located beyond the macula orbeyond the fovea so as not to block foveal perfusion. The procedure doesnot require major invasive surgery and is only minimally invasive, incontrast to procedures that involve surgical implantation of anelectrode or photovoltaic apparatus. The procedure locates particlesdiffusely substantially throughout the eye, or selected regions of theeye, in contrast to procedures in which an electrode or other device islocated at a single site. Thus, the site of treatment is expanded withthe inventive method. In this way, the particles locate within excitablecells, such as the retina, macula, etc. using an ocular example, andalso locate between these excitable cells, and are thus dispersedsubstantially throughout a region of interest. Particles not located asdescribed are handled by the retinal pigment epithelium.

Continuing to use the eye as a non-limiting example, the particlesmigrate through spaces of retinal cells and distribute through retinallayers, including the RPE. To even more widely disperse particlesthroughout the retina, they may be sprayed over the retina. In oneembodiment, they may be delivered and distributed throughout the retinallayers by a spraying or jetting technique. In this technique, apressurized fluid (liquid and/or gas) stream is directed toward atargeted body tissue or site, such as retinal tissue, with sufficientenergy such that the fluid stream is capable of penetrating the tissue,e.g., the various retinal layers. In applications, the fluid stream,which may be a biologically compatible gas or liquid, acts as a carrierfor the particles. By way of example, the spraying technique has beenused in cardiac and intravascular applications for affecting localizeddrug delivery. The teaching of those applications may be applied to thedelivery at the particles to the retina. For example, U.S. Pat. No.6,641,553 which is expressly incorporated by reference herein, disclosespressurizing a fluid carrier having a drug or agent mixed therewith andjetting the mixture into a target tissue.

It will also be appreciated that other agents may be included in thefluid in addition to the particles. These other agents include, but arenot limited to, various molecules, drugs that have stimulatory orinhibitory activity (e.g., protein drugs, antibodies, antibiotics,anti-angiogenic agents, anti-prostaglandins, anti-neoplastic agents,etc.), vectors such as plasmids, viruses, etc. containing genes,oligonucleotides, small interfering RNA (siRNA), microRNA (miRNA), etc.

In one embodiment, quantum dots conjugated or otherwise associated witha molecule or biomolecule are delivered to an eye to enhance functionalrecovery of an at least partially functional retinal cell in a patientin need of such treatment. This embodiment of the method may be inaddition to, or in place of, the method of regulating membrane polarityusing the introduced quantum dot previously described. The quantumdot-biomolecule conjugate or particle may be provided to a retinal cellcytoplasm or a retinal cell nucleus, with injection or otherintroduction means into the subretinal space, into the retina itself,into the macula, under the macula, into the vitreous cavity withvitreous fluid present, and/or into the vitreous cavity with vitreousfluid absent. The quantum dots conjugated or otherwise associated with avector carrying a protein or other molecule capable of modifying genesin retinal cell provides gene therapy. In one embodiment, racking means(e.g., sensors or other signals) associated with the complex are used tomonitor location, stability, functionality, etc. of the complex.

In one embodiment the retinal or other cell so modified by the methodcontains a light-sensitive protein that itself may be excited directlyby light of a specific wavelength, or in an alternative embodiment, beexcited by light of a different wavelength or produced by the quantumdot (e.g., fluorescence) after the quantum dot is excited upon exposureof light. For example, if the modified genes of the cell producehalorrhodopson, then the quantum dots to which thehalorhodopsin-encoding gene were associated can be excited to thenactivate the halorhodopsin to silence the cell. If the modified genes ofthe cell produce channelrhodopsin, then the quantum dots to which thechannelrhodopsin-encoding genes were associated can enhance an actionpotential. As known to one skilled in the art, channelrhodopsins, afamily of proteins, function as light-gated ion channels in controllingelectrical excitability among other functions. As known to one skilledin the art, halorhodopsin is a light-activated chloride-specific ionpump. When quantum dots are combined with channelrhodopsins orhalorrhodopsons, quantum dots enhance the effects of these proteins, andresult in enhanced cell polarization responsive to light stimulation.

In one embodiment, quantum dots conjugated or otherwise associated witha molecule or biomolecule are delivered to the heart to enhancefunctional recovery of an at least partially functional heart cell in apatient in need of such treatment.

In one embodiment of monitoring, a video camera receives an image of theexternal environment that is projected into an eye containing thefunctional, excitable retinal cell to be treated. For example, afterinitial administration of the quantum dots to the eye, a camera mountedon or in the eyeglasses records and produces a digitized image of theexternal environment, which is then transmitted to a small computermounted on the glasses. The picture can be recreated on an LCD using adiode array. This image, in turn, is projected through the pupil, ontothe retina containing quantum dots to stimulate rods and cones. Thisprocess may be optionally repeated to determine the extent or degree toexcite the quantum dots and/or to achieve the desired cell polarizationstate by evaluating retinal function, e.g., by electroretinogram orother methods known to one skilled in the art.

In one embodiment, the eye imaging method, e.g., OCT, confocalmicroscopy, provides a method of tracking the quantum dots in cells,e.g., stable cells such as neurons.

In one embodiment, the treated cells are restored to normal polarizationby treatment using the quantum dots; and concomitantly, the cells aretreated with a biological moiety conjugated to the quantum dots torelieve, restore, ameliorate, or treat a functional condition of theretinal cell, e.g., a retinal genetic disease. In one embodiment, thebiologically active conjugate is biologically active after the quantumdot ceases to be functional. In one embodiment the quantum dot is activeafter the biologically active conjugate ceases to be functional.

As schematically shown in FIGS. 3 and 4, a device 150 for delivering theparticles to the retina generally includes an elongated tube or cannula152 having a proximal end 154 and a distal end 156 and an interior lumen158 extending between the proximal and distal ends 154, 156. A distalend region 160, which may include a distal end face or a portion of theouter surface of the cannula 152 adjacent the distal end 156, includes aplurality of outlet ports or apertures 162 in fluid communication withthe interior lumen 158. The device 150 further includes a pressurecontrol source 164, such as for example a fan or pump, in fluidcommunication with the lumen 158 and operable for establishing anelevated pressure within the lumen. As known to one skilled in the art,the pressure should be sufficient to effectively disseminate theparticles throughout the retina through a spraying or jetting action,but not sufficient to substantially damage retinal tissue. In oneembodiment, a pressure may range from 0.0001 psi to 100 psi. Thepressurized spraying also assists in distributing particles thatdisseminate and localize throughout the retinal layers. Localization ofthe particles permits enhanced control, duration, ease, etc. ofstimulating these particles, resulting in enhanced control and effect.

The particles are introduced into the interior lumen 158 from anysource, such as from a reservoir chamber, a syringe, etc. (not shown),and we mixed with a carrier fluid 166 such as a biocompatible gas orliquid. As non-limiting examples, air, oxygen, nitrogen, sulfurhexafluoride other perfluorocarbon fluids, etc., alone or incombination, may be used.

The pressurized fluid carrying the particles is regulated for ejectionfrom the outlet ports, and is propelled toward the retina. The diameterof the outlet ports and pressure of the fluid are such as to allow theparticles to penetrate the retinal tissue with minimal or no retinaldamage. To accomplish a wide distribution of the particles throughoutthe retinal layers, the pressure may be pulsed to vary the penetrationdepth of the particles. The cannula may also be rotated or moved tospray or cover a larger area of the retina. Those of ordinary skill inthe art will recognize other ways to distribute the particles throughoutthe retinal layers. As one example, the diameter of the outlet ports maybe varied to provide different penetration depths. The outlet portdiameters may range from about 0.01 mm to about 1 mm. As anotherexample, the angles of the outlet ports may be varied to providedifferent spray patterns.

The above-described device may be used in the inventive method todeliver particles to the retina and distribute them substantiallythroughout the retinal layers, both intracellularly and/orintercellularly. That is, the particles diffusely locate and penetratethe retinal layers.

In one embodiment, an ocular surgeon may remove the vitreous gel, suchas by an aspiration probe having vacuum pressure or a cutting probe, andreplacing the contents of the vitreous cavity with saline, air, oranother biocompatible fluid to facilitate particle penetration. Thespraying device is inserted through the incision and into the vitreouscavity. The distal end of the device is positioned on or adjacent theretina, with the surgeon verifying placement using an operatingmicroscope, a slit lamp, or other methods known in the art. Once thedistal end of the device is adequately positioned, the pressurized fluidstream carrying the particles is generated and the particles arepropelled toward the retina so as to distribute the particles throughoutthe retinal layers, as previously described. A gas probe may also beinserted into the vitreous cavity, such as by a second incision, tomaintain the desired intraocular pressure. In another embodiment, thevitreous gel is not removed and the particles are injected (e.g., usinga needle or other type of injection device) without spraying close tothe retina, where the particles then diffuse through intercellularspaces of the retina and throughout the eye. Those of ordinary skill inthe art will recognize that while the delivery method has been describedas using separate aspiration probes, fiber optic probes, and gas probes,a single device that accomplishes delivery of the particles to theretina, removal of the vitreous gel and gas delivery may be used in theinventive method.

Once located at the desired location, the particles are stimulated usingan energy source. The energy source may be located external to the eyeat either or both the front and back, external to the retina, or on thesurface of the retina. Because the retina is transparent, light is ablepass through and hence activate the particles located on and in variousretinal tissues, as is subsequently described. The activated particlesreset or influence the plasma membrane electrical potential of excitablecells, resulting in a desired response in membrane polarity. Aspreviously described, this may take the form of normalized polarization,repolarization, enhanced polarization (i.e., stimulation), or reducedpolarization (i.e., calming), etc.

In one embodiment, the particles are delivered into the eye when thevitreous gel is removed and replaced with saline and the internallimiting membrane (ILM) is removed. In one embodiment, the internallimiting membrane is removed to permit particle dissemination within theretina and throughout retinal intracellular spaces. This enhancesdiffusion of particles in the retina so that, by fluid flow, particlescan then disseminate and penetrate retinal layers. Particles may adhereto the outer cellular membrane and/or may enter retinal cells. Theparticle size and/or spraying pressure, location, formulation may bealtered to aid in selectivity. Particle penetration may be limited bythe external limiting membrane (ELM), which may act as a semi-barrier toretinal transport. Excess particles may be removed as a part of thenormal phagocytosis process (e.g., by glial cells). Ganglial cells inthe eye, responsible for visual processing (discerning motion, depth,fine shapes, textures, colors), have less active phagocytosismechanisms, so treatment of these cells may be affected by spraying tominimize excess distribution of particles.

Repolarization of cell membranes in a first location may have beneficialeffects on polarization of cell membranes in second and subsequentlocations. Due to propagation of electrical stimuli, a wave ofelectrical distribution is disseminated throughout the retina, forexample, along a glial cell network. Because the glial cells assist inmaintaining electrical balance, propagation also stabilizes polarizationof adjacent cells.

It will be appreciated from the above description that stimulation ofthe entire retina may be achieved, rather than stimulation of a portionof the retina in proximity to a fixed electrode. This achievessubstantially uniform repolarization, minimizing or preventing areas ofhyper- and/or hypo-polarization, which assist in functional regenerationof glial cells.

In one embodiment, an ocular surgeon may stimulate the particles with anexternal light source, by ambient light, by ultrasound radiation, or byother mechanisms known to one skilled in the art. The particlesfacilitate, enhance, or boost a biological cell's regulation of itspolarity, with adjacent cells capable of being stimulated due to theglial stimulus-propagating network.

Each of the following references is expressed incorporated by referenceherein in its entirety.

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Other variations or embodiments of the invention will also be apparentto one of ordinary skill in the art from the above description. As oneexample other forms, routes, and sites of administration arecontemplated. As another example, the invention may be used in patientswho have experienced ocular trauma, retinal degeneration, ischemia,inflammation, etc. As another example, the particles may include sensingdevices for qualitative and/or quantitative chemistry or otherdeterminations. For example, the particles may include sensors or otherdetection means for glucose, oxygen, glycosylated hemoglobin, proteinsincluding but limited to enzymes, pressure, indicators for retinaldegenerative disease, etc. Thus, the forgoing embodiments are not to beconstrued as limiting the scope of this invention.

What is claimed is:
 1. A method to promote functional recovery andcontrollably regulate plasma membrane polarization of a cell, the methodcomprising providing quantum dots to an individual in need thereof totarget a cell, and applying light to the quantum dots under conditionssufficient to controllably activate the quantum dots by controlling atleast one of time of exposure, intensity of exposure, or site ofexposure, to controllably regulate the plasma membrane polarization ofthe cell and ameliorate a condition resulting from dysregulated plasmamembrane polarization of the cell.
 2. The method of claim 1 wherein theindividual has a neurological condition selected from the groupconsisting of a condition of the central nervous system, a condition ofthe peripheral nervous system, and combinations thereof.
 3. The methodof claim 1 wherein the cell is selected from the group consisting of abrain cell, a retinal cell, an olfactory cell, an auditory cell, aninferior laryngeal cell, a heart cell, and combinations thereof.
 4. Themethod of claim 1 wherein the individual has Parkinson's disease and thequantum dots are administered to regulate a dopamine-responsive neuron.5. The method of claim 1 wherein the individual has Alzheimer's diseaseand the quantum dots are administered to regulate a dopamine-responsiveneuron.
 6. The method of claim 1 wherein the individual has epilepsy. 7.The method of claim 1 performed to regulate spreading depression in theindividual.
 8. The method of claim 1 wherein the individual has aneurological condition selected from the group consisting of migraines,mood disorders, schizophrenia, stuttering, and combinations thereof. 9.The method of claim 1 where the individual in need thereof has retinitispigmentosa, retinal degeneration, posttraumatic epilepsy, restless legsyndrome, spinal cord epilepsy, Tourette's syndrome, senile dementia, ordepression.
 10. The method of claim 1 where the light applied is atleast one of ambient light, ultraviolet light, visible light, orinfrared light.
 11. A method to selectively regulate a specific nerve orcell for therapy, the method comprising providing quantum dots to anindividual at a site selected from the group consisting of a cranialnerve, a spinal cord nerve, an optic nerve, an olfactory nerve, aauditory nerve, a vocal nerve, heart muscle, and combinations thereof,and applying light to the quantum dots under conditions sufficient tocontrollably activate or de-activate an action potential in the nerve orcell using the quantum dots by controlling at least one of time of lightexposure, intensity of light exposure, or site of light exposure of thequantum dots, thereby regulating the nerve or cell and ameliorating aneurological condition.
 12. The method of claim 11 wherein the activatedquantum dots stimulate a selected area of the central nervous system.13. The method of claim 11 wherein a visual cortex is stimulated throughlight stimulation of the retina, an olfactory neuron is stimulated bysmell, an auditory neuron is stimulated by sound, and combinationsthereof.
 14. The method of claim 11 wherein the activated quantum dotsstimulate a selected area of the peripheral nervous system.
 15. Themethod of claim 11 wherein a sciatic nerve is stimulated to amelioratechronic pain, an enteric nerve is stimulated to reduce appetite, andcombinations thereof.
 16. The method of claim 11 further comprisingusing a fiber optic device surgically placed at a desired area toactivate the quantum dots.
 17. The method of claim 11 further comprisinginjecting quantum dots into a superficial area of a brain and/or nerve,and applying an appropriate wavelength of light pulse to the area toactivate the quantum dots.